Red Algal Sexual Reproduction Toxicity Tests

29 Pages·2012·2.12 MB·English

Whole effluent toxicity • training Video SerieS • Saltwater Series
Red Algal (Champia parvula)
Sexual Reproduction
Toxicity Tests
Supplement to Training Video
U.S. Environmental Protection Agency
Office of Wastewater Management
Water Permits Division
1200 Pennsylvania Ave., NW
Washington, DC 20460
EPA 833-C-09-001
March 2009
NOTICE
The revision of this guide has been funded wholly or in part by the
Environmental Protection Agency under Contract EP-C-05-063. Mention of
trade names or commercial products does not constitute endorsement or
recommendation for use.
U.S. ENVIRONMENTAL PROTECTION AGENCY Red Algal (Champia parvula) Sexual Reproduction Toxicity Tests
Supplement to Training Video
Foreword
This guide serves as a supplement to the video “Red Algal (Champia parvula) Sexual Reproduction Toxicity
Tests” (EPA, 2009). The methods illustrated in the video and described in this guide support the methods
published in the U.S. Environmental Protection Agency’s (EPA’s) Short-term Methods for Estimating the
Chronic Toxicity of Effluents and Receiving Waters to Marine and Estuarine Organisms, Third Edition (EPA,
2002a), referred to as the Saltwater Chronic Methods Manual. The video and this guide provide details on
preparing for and conducting the test based on the expertise of personnel at the following EPA Office of
Research and Development (ORD) laboratories:
National Health and Environmental Effects Research Laboratory (NHEERL) – Atlantic Ecology Division
in Narragansett, Rhode Island
NHEERL – Gulf Ecology Division in Gulf Breeze, Florida
National Exposure Research Lab (NERL) – Ecological Exposure Research Division (EERD) in
Cincinnati, Ohio
This guide and its accompanying video are part of a series of training videos produced by EPA’s Office of
Wastewater Management. This Saltwater Series includes the following videos and guides:
“Mysid (Americamysis bahia) Survival, Growth, and Fecundity Toxicity Tests”
“Culturing Americamysis bahia”
“Sperm Cell Toxicity Tests Using the Sea Urchin, Arbacia punctulata”
“Red Algal (Champia parvula) Sexual Reproduction Toxicity Tests”
“Sheepshead Minnow (Cyprinodon variegatus) and Inland Silverside (Menidia beryllina) Larval Survival
and Growth Toxicity Tests”
The Freshwater Series, released in 2006, includes the following videos and guides:
“Ceriodaphnia Survival and Reproduction Toxicity Tests”
“Culturing of Fathead Minnows (Pimephales promelas)”
“Fathead Minnow (Pimephales promelas) Larval Survival and Growth Toxicity Tests”
All of these videos are available through the National Service Center for Environmental Publications
(NSCEP) at 800 490-9198 or [email protected].
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contentS
Foreword ..................................................................................................................................................i
Introduction ............................................................................................................................................1
Background ............................................................................................................................................1
Culturing Champia parvula ....................................................................................................................1
Culture Water .........................................................................................................................................1
Photoperiod ............................................................................................................................................2
Culture Vessels ......................................................................................................................................2
Preparing Algae for Testing ...................................................................................................................2
Conducting the Test ...............................................................................................................................3
Collecting the Algae ...............................................................................................................................3
Effluent Preparation ...............................................................................................................................3
The Exposure Period ..............................................................................................................................5
The Recovery Period ..............................................................................................................................6
Terminating the Test ..............................................................................................................................6
Test Acceptability and Data Review ......................................................................................................9
Citations and Recommended References ..........................................................................................9
Glossary ..................................................................................................................................Glossary-1
Appendix A: Nutrients and Media ......................................................................................................A-1
Appendix B: Apparatus and Equipment ............................................................................................B-1
Appendix C: Reagents and Consumable Materials ...........................................................................C-1
Appendix D: Summary of Test Conditions and Test Acceptability Criteria for the Red Macroalga,
Champia parvula, Sexual Reproduction Test With Effluents and Receiving Waters .....................D-1
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U.S. ENVIRONMENTAL PROTECTION AGENCY Red Algal (Champia parvula) Sexual Reproduction Toxicity Tests
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figureS
Figure 1. Life History of the Red Macroalga, Champia parvula. Left: Size and Degree of Branching
in Female Branch Tips Used For Toxicity Tests ....................................................................................2
Figure 2. Apex of Branch of Female Plant, Showing Sterile Hairs and Reproductive Hairs
(Trichogynes) .........................................................................................................................................3
Figure 3. A Portion of the Male Thallus Showing Spermatial Sori. The Sorus Areas Are Generally
Slightly Thicker and Somewhat Lighter in Color ..................................................................................3
Figure 4. A Magnified Portion of a Spermatial Sorus. Note the Rows of Cells that Protrude from the
Thallus Surface ......................................................................................................................................4
Figure 5. Apex of a Branch on a Mature Female Plant That Was Exposed To Spermatia from a Male
Plant ........................................................................................................................................................4
Figure 6. Receiving Water Data Form for the Red Macroalga, Champia parvula, Sexual
Reproduction Test. .................................................................................................................................5
Figure 7. A Mature Cystocarp ...............................................................................................................6
Figure 8. Comparison of a Very Young Branch and an Immature Cystocarp .....................................7
Figure 9. An Aborted Cystocarp. ............................................................................................................7
Figure 10. Cystocarp Data Sheet for the Red Macroalga, Champia parvula, Sexual Reproduction
Test .........................................................................................................................................................8
tableS
Table A-1. Nutrient Stock Solution .....................................................................................................A-1
Table A-2. GP2 Artificial Seawater for Use in Conjunction with Natural Seawater for the Red
Macroalga, Champia parvula, Sexual Reproduction Toxicity Test ....................................................A-2
Table A-3. Preparation of Test Solutions at a Salinity of 30‰ Using HSB for a Final Test
Concentration Volume of 1000 mL. ...................................................................................................A-3
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U.S. ENVIRONMENTAL PROTECTION AGENCY Red Algal (Champia parvula) Sexual Reproduction Toxicity Tests
Supplement to Training Video
Introduction
This guide accompanies the Environmental Protection Agency’s (EPA’s) video training for conducting red
algal (Champia parvula) sexual reproduction toxicity tests (EPA, 2009). The test method is found in Section
16 of EPA’s Short-term Methods for Estimating the Chronic Toxicity of Effluents and Receiving Waters to
Marine and Estuarine Organisms, Third Edition (EPA, 2002a). The test was developed by EPA’s Office of
Research and Development’s (ORD’s) National Health and Environmental Effects Research Laboratory-
Atlantic Ecology Division (NHEERL-AED) in Narragansett, Rhode Island. The material presented in both the
video and this guide summarizes the methods but does not replace a thorough review and understanding
of the methods by laboratory personnel before conducting the test.
Background
Under the National Pollutant Discharge Elimination System (NPDES) program (Section 402 of the Clean
Water Act), EPA uses toxicity tests to monitor and evaluate effluents for their toxicity to biota and their
impact on receiving waters. By determining acceptable or safe concentrations for toxicants discharged
into receiving waters, EPA can establish NPDES permit limitations for toxicity. These Whole effluent toxic-
ity (WET) permit limitations regulate pollutant discharges on a whole effluent effect basis rather than by a
chemical-specific approach only.
Whole effluent toxicity methods measure the synergistic, antagonistic, and additive effects of all the chemi-
cal, physical, and additive components of an effluent that adversely affect the physiological and biochemi-
cal functions of the test organisms. Therefore, healthy organisms and correct laboratory procedures are
essential for valid test results. Laboratory personnel should be very familiar with the test methods and with
red algae handling techniques before conducting a test.
This supplemental guide covers the procedures for conducting the test according to EPA’s promulgated
methods (40 CFR Part 136; EPA, 2002c) and also provides some helpful information that is not presented
in the Saltwater Chronic Methods Manual (EPA, 2002a).
This guide summarizes methods developed at NHEERL-AED for estimating the chronic toxicity of marine
or estuarine effluents and receiving waters on the sexual reproduction of the marine macroalga, Champia
parvula. Males and females are exposed to effluents or receiving waters for 2 days, followed by a 5- to
7-day recovery period for the female plants in a control medium. Cystocarp production by the female, which
indicates sexual reproduction, is used as the endpoint. The test results determine the effluent concentra-
tion causing a statistically significant reduction in the number of cystocarps formed.
This guide and accompanying video describe how the test is set up, initiated, terminated, and reviewed,
including suggestions on maintaining healthy cultures of test organisms.
Culturing Champia parvula
There are three macroscopic stages in the life history of Champia. The adult plant body (thallus) is hollow,
septate, and highly branched. Only the mature male and female plants are used in toxicity testing. Mature
plants are illustrated in Figure 1.
To keep a constant supply of plant material available, maintain several unialgal stock cultures of males
and females simultaneously. Also, new cultures should be started weekly from excised branches so that
cultures are available in different stages of development.
culture Water
Natural seawater, or a 50-50 mixture of natural and artificial seawater, makes optimal culturing media.
Seawater for cultures is filtered at least to 0.45 µm to remove most particulates and autoclaved for 30
minutes at 15 psi (120ºC). Carbon stripping the seawater may be necessary before autoclaving to enhance
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U.S. ENVIRONMENTAL PROTECTION AGENCY Red Algal (Champia parvula) Sexual Reproduction Toxicity Tests
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its water quality (EPA, 1990). Figure 1. Life History of the Red Macroalga, Champia parvula. Left: Size
Instructions for carbon and Degree of Branching in Female Branch Tips Used For Toxicity Tests
stripping are provided in the
Saltwater Chronic Methods spermatia
Manual (EPA, 2002a). Nutrients
should be added to the water
to ensure healthy cultures.
MALE
Recipes for the culturing
meiosis
medium and nutrient solutions
are provided in Appendix A. The
fertilization
water temperature should be
maintained at 23ºC ± 1ºC and
the salinity at 30‰ ± 2‰ tetrasporangia
Gently aerate the cultures.
TETRASPOROPHYTE
Change alternate cultures’
FEMALE cystocarp
medium every week so that if a
stock solution should become 5 mm
contaminated, the entire batch Source: EPA 1987.
will not be lost. While replen-
ishing the medium, divide the growing algae in half with sharp forceps or discard half of the biomass to prevent
overcrowding. New cultures also can be started at this time using 1 cm branch tips. Add nutrients using a pipet;
NHEERL-AED has found a squeeze bottle is quick and easy to use. At the end of approximately three weeks, there
should be enough plant material to conduct the test.
PhotoPeriod
The culture conditions should include a photoperiod 16 hours of light and 8 hours of darkness. The light
level should not exceed 500 ft-candles (75 µE/m2/s) and may have to be adjusted to that level, depending
on the reflecting characteristics of the incubators.
culture VeSSelS
Maintain stock cultures of males and females in separate, aerated, 1 L Erlenmeyer flasks containing 800
mL of the culture medium. All glass must be acid-stripped in 15 percent HCl and rinsed in deionized water
before use because some detergent residues can be toxic to the Champia. At least every 6 months, the
glass should be cleaned to remove organic materials that can build up on the surface. Always use sterile
techniques when culturing the algae (i.e., autoclave all stock solutions and flame all tools before cutting or
transferring plants) to guard against microalgal contamination.
PreParing algae for teSting
Examine the stock cultures to determine their readiness for testing. Place a few female branch tips in
seawater in a petri dish, and examine them under a compound microscope to determine if trichogynes are
present. An inverted scope works best with the petri dishes, although standard slides and microscopes also
can be used. Trichogynes are the short, fine reproductive hairs to which the spermatia attach (see Figure
2). They should be seen easily near the apex of the branch tip. Although both sterile hairs and trichogynes
occur on the apex, sterile hairs occur over the entire plant thallus. Sterile hairs are wider and generally much
longer than trichogynes, and appear hollow, except at their tip, where they seem to be plugged.
Males should be visibly producing spermatia. Sometimes, the presence of spermatia sori can be deter-
mined by placing some male tissue in a petri dish and holding it against a dark background. Mature sori
can be easily identified under a microscope along the edge of the thallus. The sorus areas are generally
thicker and lighter in color than the rest of the plant body. At higher magnification, the spermatia them-
selves can be seen (see Figures 3 and 4).
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U.S. ENVIRONMENTAL PROTECTION AGENCY Red Algal (Champia parvula) Sexual Reproduction Toxicity Tests
Supplement to Training Video
The readiness of the male stock Figure 2. Apex of Branch of Female Plant, Showing Sterile Hairs and
culture can also be assessed Reproductive Hairs (Trichogynes)
by placing a portion of a female
sterile hairs
plant into a portion of the solu-
tion from the male culture for
a few seconds. Under a micro-
scope, numerous spermatia
should be seen attached to the
sterile hairs and trichogynes of
the female plant (see Figure 5).
trichogynes
Once readiness is established
for both males and females, the
test can begin.
Conducting the Test
collecting the algae
Prepare cuttings from the most 1 mm
healthy-looking plants. Prepare
the female cuttings first to mini-
Sterile hairs are wider and generally much longer than trichogynes, and appear hollow
mize the chances of contaminat- except at the tip. Both types of hairs occur on the entire circumference of the thallus, but
ing them with water containing are seen easiest at the “edges.” Receptive trichogynes occur only near the branch tips.
spermatia from the male stock
Source: EPA 1987.
cultures. Place each plant in
a petri dish containing a small amount of seawater. Using a fine-point forceps or scalpel, prepare five cut-
tings from the female plants for each treatment replicate, severing the plant 7 – 10 mm from the ends of the
branch. Try to be consistent in the degree of branching in the cuttings, since cystocarps form at the branch
tips.
For male plants, use one cutting for each treatment replicate, severing the plant about 2 – 3 cm from the
end of the branch. If there are few branches, or the spermatial sori appear sparse, larger male cuttings may
be needed. The cuttings can be kept at room temperature for up to an hour.
effluent PreParation Figure 3. A Portion of the Male Thallus Showing Spermatial Sori. The
Sorus Areas Are Generally Slightly Thicker and Somewhat Lighter in
Effluent sampling should be
Color
conducted according to Section
8 of the Saltwater Chronic
Methods Manual (EPA, 2002a)
and any specific requirements of
a NPDES permit. The effluent or 1 cm
receiving waters should be held
at 0ºC – 6ºC until used for test-
ing. Under the NPDES program,
spermatial sorus
lapsed time from sample collec-
tion to first use in the test must
not exceed 36 hours. Under
special conditions or variances,
samples may be held longer but
should never be used for testing
if held for more than 72 hours.
Source: EPA 1987.
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U.S. ENVIRONMENTAL PROTECTION AGENCY Red Algal (Champia parvula) Sexual Reproduction Toxicity Tests
Supplement to Training Video
Figure 4. A Magnified Portion of a Spermatial Sorus. Note the Rows
Dilution Water
of Cells that Protrude from the Thallus Surface
The type of dilution water used
to make the test concentrations
is dependent on the objectives
cuticle
of the test. Any specific require-
ments included in NPDES permits
should be followed. The Saltwater
Chronic Methods Manual (Section
7) provides the following guidelines:
• If the test is conducted to esti-
spermatia
mate the absolute chronic tox-
icity of the effluent, synthetic 100 µm
dilution water should be used. If
the cultures were maintained in
different water than used for dilu- thallus surface
tion water, a second set of control
replicates should be conducted
Source: EPA 1987.
using the culture water.
• If the test is conducted to
estimate the chronic toxicity
of the effluent in uncon- Figure 5. Apex of a Branch on a Mature Female Plant That Was
taminated receiving waters, Exposed To Spermatia from a Male Plant
the test can be conducted using
a grab sample of the receiving
waters collected outside the influ-
spermatia
ence of the outfall, other uncon-
taminated waters, or standard
dilution water with the same
salinity as the receiving waters. If
the cultures were maintained in
different water than used for dilu-
tion water, a second set of control
replicates should be conducted
using the culture water.
• If the test is conducted to
estimate the additive or miti-
gating effects of the effluent
The sterile hairs and trichogynes are covered with spermatia. Note that
on already contaminated
few or no spermatia are attached to the older hairs (those more than
receiving waters, the test 1 mm from the apex).
must be conducted using receiv- Source: EPA 1987.
ing waters collected outside the
influence of the outfall. Controls
should be conducted using both
receiving water and culture water.
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